The ability of HIV to enter a latent state creates a fundamental obstacle to the eradication of HIV-1 infection by anti-retroviral therapy and has prompted renewed interest in developing inhibitors that block the re-emergence of latent proviruses. The goal of this research project is to discover new antiviral drugs that target the essential interaction between the Human Immunodeficiency Virus (HIV) Tat protein and TAR RNA, which is required to positively regulate transcriptional elongation from the HIV-1 promoter. An inhibitor of the Tat-TAR interaction would be able to block viral replication both in acutely and in chronically infected cells and selectively target the reservoir of slowly replicating viruses that persists in the presence of Highly Active Anti Retroviral Therapy (HAART). In the previous funding period, we utilized a peptidomimetic approach grounded in structure-based design to discover potent inhibitors of the Tat-TAR interaction. The lead peptidomimetic compounds penetrate cellular membranes, inhibit Tat-dependent transactivation in human cells and inhibit viral replication in primary lymphocytes for a variety of strains through a Tat-dependent mechanism. We now propose to: 1. Optimize the activity of the cyclic peptide mimetics of the Tat protein to inhibit the HIV-1 Tat-TAR interaction and Tat activity in cell extracts 2. Extend the peptide to additionally target the interaction of cyclin T1 with the apical loop of TAR RNA, thereby preventing formation of the Tat-TAR-cycT1 ternary complex and recruitment of the Ptef-b kinase by two mechanisms simultaneously 3. Optimize the uptake and cellular localization of the peptidomimetics and evaluate the activity of the lead compounds in functional cell-based assays 4. Evaluate the activity of the lead compounds in inhibiting viral replication and study the emergence of resistance in the virus in the presence of the peptidomimetic inhibitors. The project will provide the information required to guide the pharmacological development of these compounds. We are very confident that highly selective compounds with ICSOs well below 100 nM can be developed in the course of this program.